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Spus et al. Microbiome Res Rep 2023;2:33 https://dx.doi.org/10.20517/mrr.2023.20 Page 5 of 15

Figure 1. Design of the multi-strain blend used in the propagation experiment. Lactococcus lactis, Lactococcus cremoris and Leuconostoc
mesenteroides were represented with 24 strains from eight genetic lineages of the Ur starter culture. The 24 strains are visualized in the
first (inner) circle of the diagram where each color indicates a different genetic lineage. For bacteriophage challenge, a cocktail of three
bacteriophages was used including lactococcal phage ϕTIFN1, ϕTIFN5 and ϕTIFN7. Strains in the blend differ among each other in terms
of resistance to phages present in the cocktail - illustrated by black, dark grey and light grey columns in the second (outer) circle of the
diagram. Lineage 8 encompasses Lc. mesenteroides ssp. cremoris strains, which are resistant to lactococcal phages used in this study. Cit:
Citrate degradation ability; Lac: lactose utilization ability; Prt: caseinolytic protease activity.

Phage-resistance assay
To test if the strains used in blends evolved resistance or to screen for possibly induced prophages during
[27]
the course of the experiment, we used a qualitative spot assay . The supernatants of the blends at different
time points (after 6.6, 139, 272, 405, and 538 generations) were used as a possible source of phages and
spotted onto the bacterial cell lawn of each of the strains used in the blend preparation. In detail, 10 µL of
filtered supernatant (0.2 µm pore size sterile Minisart® filters, Sartorius Stendim Biotech, Göttingen,
Germany) was used to spot each indicator culture in the soft agar layer (0.75% agar w/v; 0.5% lactose;
10 mM CaCl ).
2

The same procedure was performed with the end time point (after 538 generations) isolates (200 single
colony isolates, 50 isolates per replicate) using the filtered supernatant as well as the undiluted solution
(10 pfu/mL) of all three bacteriophages used in the cocktail (ϕTIFN1, ϕTIFN5, and ϕTIFN7). To obtain
10
single colony isolates from the propagated cultures, all four blends were diluted in PPS and plated onto
LM17 agar. After 48 h of incubation at 30 °C, 50 single colonies of each blend were picked from the plates,
transferred to LM17 broth, and incubated for 24 h. These new single colony isolates were preserved at
-80 °C in glycerol (20% v/v) for further use.